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Home , Chromatophore, Doryteuthis opalescens

© 2017. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2017) 220, 4669-4680 doi:10.1242/jeb.164160

RESEARCH ARTICLE Myogenic activity and serotonergic inhibition in the network of the Dosidicus gigas (family Ommastrephidae) and opalescens (family ) Hannah E. Rosen* and William F. Gilly

ABSTRACT Chromatophore displays of living squid (chromogenic behaviors) Seemingly chaotic waves of spontaneous chromatophore activity have been described for other families (Moynihan, 1983a,b; Bush occur in the ommastrephid squid Dosidicus gigas in the living state et al., 2009; Burford et al., 2014; Rosen et al., 2015), but and immediately after surgical disruption of all known inputs from the comparative work on anatomy and control mechanisms is lacking. central nervous system. Similar activity is apparent in the loliginid Chromogenic behaviors are subject to strong descending motor , but only after chronic denervation of control, but coordinated chromatophore activity also occurs when ‘ ’ for 5–7 days. Electrically stimulated, neurally central control is absent, most notably in the wandering clouds ‘ ’ driven activity in intact individuals of both species is blocked by display ( wolkenwandern of Hofmann, 1907) that is readily tetrodotoxin (TTX), but TTX has no effect on spontaneous wave observed in chronically denervated of squid and activity in either D. gigas or denervated D. opalescens. Spontaneous (Packard, 1992a,b, 1995a,b). Post-denervation activity of this sort TTX-resistant activity of this sort is therefore likely myogenic, and has been studied in the loliginid squid Loligo vulgaris (Packard, such activity is eliminated in both preparations by serotonin (5-HT), a 2001, 2006, 2011) and Doryteuthis opalescens (Packard, 1995a). known inhibitor of chromatophore activity. Immunohistochemical Although waves of chromatophore activity have not been described techniques reveal that individual axons containing L-glutamate or in intact loliginids, wave-like activity is prominent in many species 5-HT (and possibly both in a minority of processes) are associated of and (Hanlon and Messenger, 1996) as well as with radial muscle fibers of chromatophores in intact individuals of in conjunction flickering in the oceanic squid Dosidicus gigas both species, although the area of contact between both types of (Rosen et al., 2015). axons and muscle fibers is much smaller in D. gigas. Glutamatergic Coupling of chromatophores within the network itself has been ‘ ’ and serotonergic axons degenerate completely following denervation proposed to permit peripheral horizontal control over myogenic – in D. opalescens. Spontaneous waves of chromatophore activity in activity in addition to the more widely recognized, descending ‘ ’ both species are thus associated with reduced (or no) serotonergic vertical control by the nervous system (Packard, 2001). input in comparison to the situation in intact D. opalescens. Such Mechanistic details of how these two control systems might differences in the level of serotonergic inhibition are consistent with actively work in concert to coordinate chromogenic behaviors are natural chromogenic behaviors in these species. Our findings also largely unknown (Packard, 2011). Vertical control is hierarchical suggest that such activity might propagate via the branching distal and top-down. Horizontal coupling is a network property, and ends of radial muscle fibers. connections between individual chromatophores must occur at the cellular level – yet little experimental attention has been paid to KEY WORDS: Squid, Chromatophore, Excitability, Denervation, relevant mechanisms or structures underlying coupling in any Serotonin, Ommastrephid species. Wave-like patterning does not have identical features in different coleoid taxa, as suggested by differences in the wandering INTRODUCTION clouds display cited above versus the repetitive ‘passing clouds’ of Chromatophores are responsible for colorful and dynamic cuttlefish (Laan et al., 2014), and the balance of control between patterning of the skin in coleoid , a subclass that central and peripheral processes in vivo, as well as basic includes squid, octopus and cuttlefish. Chromatophores in these mechanisms themselves, may differ. This paper focuses on the taxa are unique in that they are composed of an elastic sac chromatophore network in squid. surrounded by a ring of radial muscle fibers that shorten when Because the chromatophore is the effector organ for chromogenic excited, thereby expanding the pigment sac and making it visible. behaviors, regardless of the control pathways involved, properties of Although all species of squid have chromatophores, our radial muscle fibers and the axons that innervate them are important understanding of the mechanisms that control activity in the to consider. Synaptic excitation generates post-synaptic potentials chromatophore network of this group derives from studies on a in radial muscle fibers of D. opalescens and ‘graded spike potentials’ handful of species in the family Loliginidae (Messenger, 2001). that are thought to involve voltage-gated calcium channels occur in fibers of spontaneously pulsating chromatophores (Florey and Kreibel, 1969). Such graded Ca2+-based excitability is a common Department of Biology, Hopkins Marine Station of Stanford University, 120 Oceanview Blvd, Pacific Grove, CA 93950, USA. feature in muscle cells (Zachar, 1971; Schwartz and Stühmer, 1984), but comparative work on radial muscle fibers in *Author for correspondence ([email protected]) other teuthid species has not been reported. H.E.R., 0000-0002-4500-4406 Endogenous of the chromatophore network have been examined in several loliginids (Messenger, 2001); again,

Received 4 June 2017; Accepted 18 October 2017 comparative data are not available. Immunohistochemistry has Journal of Experimental Biology

4669 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 4669-4680 doi:10.1242/jeb.164160

onshore laboratory where they were maintained for up to 24 h in a List of abbreviations round tank (1.5×1 m) with recirculating seawater at 19–20°C. Squid 5-HT serotonin were killed via rapid decapitation. L-Glu L-glutamate Small specimens of D. opalescens (Berry 1911) (8–9 cm ML) ML length were caught in Monterey Bay, California, from the Monterey public ROI region of interest wharf (36.604N, 121.889W), and larger squid (13–15 cm ML) were TTX tetrodotoxin caught within 1 km of the shoreline near Hopkins Marine Station, CA, USA. All squid were caught using P-Line Squid Slayer Finesse (G. Pucci and Sons, Inc., Brisbane, CA, USA) squid jigs. revealed that L-glutamate (L-Glu) is contained in axons associated were maintained in holding tanks at Hopkins Marine Station with with radial muscle fibers (Messenger et al., 1997), application of L- flow-through seawater at ambient temperature (12–15°C). Within Glu results in tonic expansion of chromatophores (Bone and 24 h of capture, squid were anesthetized in seawater containing 1% Howarth, 1980; Florey et al., 1985; Messenger et al., 1997), and ethanol, and denervation was carried out by transecting the left iontophoretic delivery of L-Glu results in excitatory post-synaptic pallial nerve just before its entry into the stellate ganglion. This was currents in isolated radial muscle fibers (Lima et al., 2003). This done through the mantle without any other surgery. Squid body of evidence has led to the acceptance of L-Glu as the excitatory were then returned to the holding tank for 7 days and regularly fed in the chromatophore network in loliginids. live or fathead minnows in accordance with Stanford Serotonin [5-hydroxytryptamine (5-HT)] is also found in a subset University’s Institutional Care and Use Committee of axons that are apposed to radial muscle fibers in loliginid squid (IACUC) -animal protocol. After visual confirmation of (Messenger et al., 1997). Topical application of 5-HT results in spontaneous wave activity in the denervated area, the squid was retraction of expanded chromatophores (Florey, 1966; Florey and killed by decapitation for physiological and anatomical studies. Kreibel, 1969; Messenger et al., 1997) but has relatively minor effects on nerve-induced contraction/relaxation cycles (Florey and Recording of chromatophore activity Kreibel, 1969). Inhibitory postsynaptic potentials have not been For pharmacological studies with D. gigas, an entire fin from small observed in radial muscle fibers, nor does 5-HT affect membrane squid or a piece (2×2 cm) of fin from larger squid was removed and potential or ionic permeability of the muscle fibers, consistent with pinned out, ventral side up, on the Sylgard (Dow Corning, Midland, the idea that it does not act as a classical inhibitory transmitter MI, USA) bottom of a dish filled with filtered seawater at room (Florey and Kreibel, 1969). These observations, along with the temperature (∼24°C). The epidermis was left intact because it was complex nature of the intracellular, G protein-coupled pathways that not possible to remove it without damaging the underlying mediate effects of 5-HT in other systems (Hannon and Hoyer, chromatophores. For studies of (denervated) D. opalescens,a 2008), make it reasonable to conclude that 5-HT acts as an piece of mantle (2×2 cm) with skin attached and containing equal inhibitory neuromodulator in the chromatophore network of portions of the denervated area and contralateral intact side was loliginid squid. pinned out in a dish filled with filtered seawater at room temperature This paper provides the first characterization of the (20–22°C), and a small window (5×5 mm) of epidermis was chromatophore network in an ommastrephid squid, D. gigas, removed in the center of both denervated and intact portions and compares waves of chromatophore activity with those in to facilitate penetration of bath-applied reagents (TTX, 5-HT and denervated D. opalescens. Activity in both cases is resistant to L-Glu). tetrodotoxin (TTX) and eliminated by 5-HT. Waves thus occur in For both species, chromatophore activity was recorded at the absence of neuronal control through any known pathway. 30 frames s–1 (720×480 pixels) with a Sony camcorder mounted Immunohistochemical data confirm that L-Glu and 5-HT are on a tripod. Spontaneous activity was recorded for 2 min, and then endogenous to the chromatophore networks of both species. Both electrical stimuli (1 ms duration) were applied to the skin using a innervations in D. opalescens degenerate completely after tungsten bipolar electrode of 0.5 mm diameter (Rhodes Medical denervation, and both are less developed in D. gigas.We Instruments, Woodland Hills, CA, USA) and a Grass SD-9 propose that myogenic wave activity in these cases is facilitated stimulator (Grass Technologies, Quincy, MA, USA). After by weak (or no) inhibitory control exerted by 5-HT, relative to the identifying threshold, chromatophore activity for each stimulus strong control in intact D. opalescens. Putative connections was recorded for five additional stimuli increasing at 1 V observed between branching distal ends of radial muscle fibers increments. This tended to give a near maximal response for the of neighboring chromatophores suggest that these structures may strongest stimulus. The same stimuli were then delivered in be relevant to the propagation of such activity. descending order. Each voltage was delivered a total of five times, alternating between ascending and descending order. The procedure MATERIALS AND METHODS was then repeated at four additional spots on the 2×2 cm skin Animals sample (D. gigas) or 5×5 mm window (D. opalescens), one at each Dosidicus gigas (d’Orbigny 1835) was captured in the Gulf of remaining corner and one in the center. California within 3 km of Santa Rosalia, Baja California Sur, Following a stimulation experiment, the seawater was removed (27.339N, 112.267W) during June and July, 2015. All from the dish and replaced with an equal volume of test solution specimens were of the small-size-at-maturity phenotype that has containing varying concentrations of 5-HT creatinine (H7752, persisted in this area since 2010 (Hoving et al., 2013; Robinson Sigma-Aldrich, St Louis, MO, USA), 200 µmol l−1 (D. gigas)or et al., 2016). Squid with a mantle length (ML) of 14–23 cm were 100 µmol l−1 (D. opalescens) L-glutamic acid (G1626, Sigma- caught using light fishing line and small jigs, and smaller Aldrich), or 200 nmol l−1 (D. gigas) or 400 nmol l−1 (D. opalescens) individuals (8–10 cm ML) were caught at the surface with a dip TTX (1069, Tocris, Bristol, UK). These concentrations of 5-HT and net. Captured squid were immediately placed in coolers filled with L-Glu were chosen based either on published studies (Florey and ambient seawater (28–30°C) on the vessel and transported to an Kreibel, 1969; Cornwell and Messenger, 1995; Messenger et al., Journal of Experimental Biology

4670 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 4669-4680 doi:10.1242/jeb.164160

1997) or, in the case of L-Glu and TTX, by adjusting concentrations 0.5×0.5 cm pieces were taken from the center of each sample until an effective dosage was found. The stimulation procedure from both the intact and denervated sides of the fixed tissue to avoid described above was then repeated. In the case of TTX, 10–40 min any damaged edges. were allowed between the time of application and experimentation for All fixed samples were washed once in seawater, then in seawater the toxin to show a maximal effect. In some cases the test solution was mixed 1:1 with phosphate-buffered saline (PBS), and finally in PBS −1 −1 −1 removed from the dish after the stimulation procedure and replaced (10 mmol l Na2HPO4, 137 mmol l NaCl, 1.5 mmol l KH2PO4, with seawater to verify reversibility. 5 mmol l−1 KCl). Samples were stored at 4°C in PBS with 0.05% sodium azide for a maximum of 4 days. Analysis of video data Prior to carrying out antibody labeling procedures, all samples Recorded video clips were converted to image stacks using were incubated in 5 mg ml−1 type 1 collagenase (17100, Gibco, VirtualDub (v1.9.11, Avery Lee, Cambridge, MA, USA) and the Carlsbad, CA, USA) in PBS for 30 min at 37°C. Samples were then images were analyzed using MATLAB R2014b (MathWorks, blocked overnight in PBS with 0.1% Triton X-100, 6.25% goat serum Natick, MA, USA). In both species, analysis was conducted over a (G9023, Sigma-Aldrich) and 0.1% bovine serum albumin 5×5 mm square. In D. gigas, the region of interest (ROI) was (15561020, Invitrogen, Carlsbad, CA, USA) prior to and during centered on the electrode for analysis of stimulated activity. The incubation in primary antibodies to minimize nonspecific labeling. ROI for spontaneous activity was selected as the area with the most Samples fixed in paraformaldehyde only were labeled with a frequent activity (as judged visually) when the tissue was in polyclonal anti-5-HT antibody (raised in rabbit) at a dilution of seawater. This same ROI, which was assumed to be representative, 1:200 (S5545, Sigma-Aldrich) for 3 h at room temperature. Samples was used when the skin sample was tested in the experimental fixed in paraformaldehyde plus glutaraldehyde were simultaneously solution. In D. opalescens, the ROI was always the 5×5 mm2 labeled with anti-5-HT at a dilution of 1:500 and a monoclonal anti- window lacking the epidermis. glutamate antibody (raised in mouse) at a dilution of 1:10,000 The RGB values for all pixels within the ROI were summed for (G9282, Sigma-Aldrich) for 3 h at room temperature. Samples were each frame throughout the time course of the response that then washed in PBS with 0.1% Triton X-100, 6.25% goat serum and accompanied the strongest electrical stimuli (i.e. 5 V above 0.1% bovine serum albumin, and incubated for 2 h in the secondary threshold). Summed pixel values were used as a proxy for antibodies at a dilution of 1:250. Secondary antibodies used were chromatophore activity, with darker pixels of a higher value anti-rabbit with a 546 nm fluorophore (A11010, Life Technologies, representing expanded chromatophores. A 25 mm2 area was equal Carlsbad, CA, USA) to target anti-5-HT primary antibodies and anti- to roughly 100×100 pixels, yielding a maximum pixel value of mouse with a 647 nm fluorophore (A21235, Life Technologies) to 3×255×104, or 7.65×106. In order to control for inherent variation of target anti-L-Glu antibodies. chromatophore response within the same field, an average of the five Incubation with secondary antibodies also contained Alexa- responses for each spot was treated as one replicate. These replicates Fluor-488–phalloidin (A12379, Life Technologies) at a 1:125 were averaged to yield a canonical control (mean±s.d., total n=25) dilution to label actin filaments. After labeling with the secondary response for that preparation that could be compared with a test antibodies and phalloidin, the samples were incubated in 1:50 DAPI response computed in the identical manner under different (D1306, Life Technologies) for 5 min and mounted on glass slides experimental conditions. Spontaneous activity was analyzed over in 100% glycerol. a 30 s time course, and summed pixel values for the ROI were Controls were performed by incubating samples in secondary computed for each frame to define the time course of the activity. antibodies without first exposing them to any primary antibodies. No The summed variance of pixel values over the 30 s sampling period labeling was observed in such experiments. Controls for double- was used as a measurement of spontaneous transient activity. labeling experiments were carried out by labeling with one antigen at a time (Wessendorf and Elde, 1985), and no change in individual Immunohistochemistry labeling patterns was noted in the double-labeling experiments. Both Skin samples from D. gigas were prepared for immunohistochemistry primary antibodies used are commercially produced and specificities by first separating the skin from the ventral side of a fin from the are confirmed by the manufacturers. In addition, specificity of the underlying muscle. Skin samples were then pinned out at their natural anti-L-Glu antibody has been confirmed in several peer-reviewed size in filtered seawater. The iridophore layer was removed by publications, one of which compared the labeling patterns produced dissection, and the combined chromatophore layer and epidermis were by different anti-glutamate antibodies (Kolodziejczyk et al., 2008). then fixed for 4 h at room temperature in either 4% paraformaldehyde Specificity of the anti-5-HT antibody was also checked by mixing the or 4% paraformaldehyde plus 0.1% glutaraldehyde in sterile-filtered antiserum with 500 µmol l−1 5-HT creatinine overnight and then seawater. Addition of this low concentration of glutaraldehyde was using these ‘blocked’ antibodies to incubate samples from D. found to be necessary for successful glutamate labeling, but higher opalescens to be labeled as described above. This procedure greatly concentrations interfered with detection of 5-HT in double-labeling reduced 5-HT labeling in axons of the chromatophore layer. experiments due to an increase in non-specific background labeling. For immunohistochemistry in D. opalescens, a 2×3 cm piece of Imaging and analysis skin centered on the dorsal midline was removed from the mantle, Imaging of skin samples employed a Zeiss LSM 700 confocal thereby yielding two samples of both denervated and intact skin, microscope with a 20× dry objective and a 40× oil objective, and all each 1.0×1.5 cm. The intact side provided an internal control for the images were taken from areas with minimal chromatophore damage. effects of denervation in that animal. The epidermis and iridophore Under the 20× objective, tile scans were taken with each tile layers of the skin were removed by dissection, leaving only the covering a 0.32×0.32 mm area in a z-stack with a thickness of 23 µm chromatophore layer. Samples were either fixed for 2 h in 4% that spanned the entire chromatophore layer. Individual tiles for paraformaldehyde in filtered seawater at room temperature to be each layer of the z-stack were stitched together during processing to labeled for 5-HT, or for 4 h in 4% paraformaldehyde plus 0.1% create an image representing a 1.6×1.6 mm region. Scans using the glutaraldehyde to be labeled for L-Glu. After fixation, three 40× objective covered an area of 0.16×0.16 mm. Journal of Experimental Biology

4671 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 4669-4680 doi:10.1242/jeb.164160

Stereology was carried out manually on the 1.6×1.6 mm region organisms (Packard, 1995c) (Fig. 1A). Although all stitched together from tiles taken using the 20× objective in order to chromatophores of D. gigas are reddish , subtle differences quantify several features. All radial muscle fibers labeled with in skin color are evident. For example, the dorsal surface of the phalloidin in each image stack were counted. A muscle fiber was mantle shortly after death typically has a purplish , when most counted only if the proximal wedge that contained the nucleus could chromatophores are expanded (Fig. 1B), whereas the ventral side be identified and if the fiber was longer than 20 µm. Another count tends to be lighter with an tint (Fig. 1C). Such differences was made of the length of contact between muscle fibers and are at least partially due to the degree of chromatophore expansion associated antibody-labeled axons. When determining the effects of and/or different size classes of chromatophores, but these features denervation in D. opalescens, a muscle fiber with an associated cannot be easily distinguished in chromatophores of one color axon was defined by a continuous line of positive labeling of the (insets in Fig. 1B,C). In addition, numerous iridophores confer a selected neurotransmitter that maintained direct contact with a radial golden metallic-like sheen to the ventral (Fig. 1C) and lateral muscle fiber for more than 10 µm. From these measurements, the mantle. Differences between the dorsal and ventral surfaces of the percentage of radial muscle fibers with associated axons was fins, which contain few iridophores, are much less noticeable calculated. (compare Fig. 1B and C). Chromatophore density on the ventral Two muscle fibers were considered to be ‘connected’ at the distal surface of the fin (1344±273 cm−2, n=4; this study) is comparable to ends if both muscle fibers could be traced over their entire length that on the dorsal surface of the mantle (1480±174 cm−2) or head from the proximal nucleated ends to the distal connection point with (1332±155 cm−2) (Rosen et al., 2015). no detectable gap in the phalloidin labeling. Muscle fibers that simply overlapped were excluded by observing the samples on Stimulated and spontaneous chromatophore activity multiple planes using a z-stack with a vertical resolution of 3 µm Electrical stimulation with a single brief shock can elicit two types using a 20× objective. From these measurements, the percentage of of chromatophore activity in D. gigas: a rapid, localized response radial muscle fibers with putative connections to other muscle fibers and, in approximately 12% of stimulations (29/243), a delayed wave was calculated. that propagates outward from the stimulated area. The localized response is characterized by a brief latency (<33 ms=1 frame) and RESULTS the synchronous expansion of a field of chromatophores around the Chromatophore network in D. gigas site of stimulation ranging in size from a few units to an area >1 cm When alive, D. gigas typically displays a ‘resting’ coloration of in diameter (Fig. 2A). Regardless of the size of the stimulated field, reddish brown on the dorsal surface and paler red to on the peak expansion of responsive chromatophores generally occurs ventral surface, a common pattern in pelagic within 200 ms and relaxation is fairly complete within 1–2 s. This

A Fig. 1. Skin color in living Dosidicus gigas and shortly after death. (A) Color of the dorsal surface of a live squid is generally a reddish-brown that gradually fades to a lighter shade or even white on the ventral surface. (B) Color of the dorsal mantle shortly after death tends to be dark-purple–red. (C) Color of the ventral mantle of the same squid is a lighter orange– 5 cm red. Individual chromatophores can be seen in the insets. The squid pictured in panel A was B C photographed in the lab in Santa Rosalia, Baja California Sur, Mexico. The squid in panels B,C was photographed on an oceanographic cruise by RV Puma in the Gulf of California as part of an independent sampling program.

5 cm 5 cm

1 cm 1 cm Journal of Experimental Biology

4672 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 4669-4680 doi:10.1242/jeb.164160 type of response presumably results from electrical stimulation of described in the preceding paragraph, and it is likely that a common motor axons in the skin that innervate a defined field of radial mechanism underlies propagating activity in both cases. muscle fibers. Spontaneous waves similar to those described above were also In approximately 12% of the applied stimulations, the localized observed in denervated skin samples from D. opalescens (Movie 2), response was immediately followed by a propagating wave of but this activity never propagated into the contralateral control side activity that spread from the excited area at a rate of ∼0.9 cm s−1 of the same sample. Similar activity was also evident in the (Fig. 2B). The direction of propagation was typically not denervated field of the living animal (not illustrated), but activity of symmetrical with respect to the localized response. No pattern of this sort does not occur in intact living D. opalescens or in fresh skin stimulation could be found that reliably produced waves, and samples from intact squid. identical stimuli at the same spot were also inconsistent. Relaxation time of chromatophores participating in a wave is 0.5–1.5 s, slightly Pharmacology faster than for chromatophores involved in the localized response. Stimulated localized responses in both D. gigas (Fig. 3A) and intact Propagating waves also occur spontaneously in fresh skin D. opalescens (Fig. 3B) are greatly reduced by exposure to 200– preparations from D. gigas, with activity showing an irregular 400 nmol l−1 TTX. Because TTX has no known action other than pattern with the wave front spreading with a velocity of ∼0.9 cm s−1 blocking voltage-gated sodium channels, these results are consistent (Fig. 2C, Movie 1). In some cases, similar waves repeat at irregular with the idea that electrical stimulation of the skin excites branches intervals of several seconds, but the exact pattern is generally of motor axons. In contrast, the amplitude of spontaneous wave variable. Based on the nature and speed of propagation, this activity is not diminished by TTX in either D. gigas (Fig. 3C) or spontaneous wave activity is similar to the stimulated waves denervated D. opalescens (Fig. 3D).

A 1 cm

0 ms 200 ms 400 ms 600 ms B

0 ms 200 ms 400 ms 600 ms C

0 ms 200 ms 400 ms 600 ms

Fig. 2. Chromatophore activity observed in fresh skin preparations from D. gigas. (A) Electrical stimulation elicits a rapid, twitch-like response from a field of chromatophores generally localized around the point of stimulation. The left-most panel shows the skin at the time of stimulation (0 ms). Responsive chromatophores within a defined field (solid border) reach peak expansion by 200 ms and remain expanded for more than 400 ms (fourth panel). (B) On some occasions, electrical stimulation evokes a localized response (solid border, 200 ms panel) followed by a wave of chromatophore expansion that propagates at a velocity of 0.9 cm s−1 in an unpredictable direction (dashed border in 400 ms panel and dotted in 600 ms panel). These propagating responses begin immediately upon the initial response reaching its maximal amplitude (200 ms) and may be initiated by it. (C) Propagating waves similar to those evoked by electrical stimulation can also occur in the absence of stimulation. Activity spreads outward from a plaque of 10 spontaneously active chromatophores (0 ms, indicated by *) to a small area (200 ms) and then to a larger area at a velocity of 0.9 cm s−1 (dashed border at 400 ms and dotted border at 600 ms). Note that chromatophores of different size classes may contribute to the wave, but we have not studied this feature in detail. Journal of Experimental Biology

4673 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 4669-4680 doi:10.1242/jeb.164160

ABAB

Pixel value 100,000 Pixel Pixel value 50 ms value Pixel 20,000 value 60,000 30,000 50 ms 50 ms 50 ms C C

Pixel value 200,000 Pixel value 200,000 2 s 2 s

D D

Pixel value 500,000 Pixel value 500,000 2 s

2 s Fig. 3. Effects of tetrodotoxin (TTX) on chromatophore activity. Stimulated activity is greatly suppressed by TTX in both species, but amplitude of Fig. 4. Effects of serotonin (5-HT) on chromatophore activity. Stimulated spontaneous wave activity is not affected. (A) Mean chromatophore response responses are greatly reduced by 2.5 µmol l−1 5-HT in D. gigas but not in (±s.e.m.) in a fresh skin preparation from D. gigas evoked by electrical D. opalescens, whereas 5-HT eliminates wave activity in both intact D. gigas stimulation 5 V above threshold (n=20). symbols represent responses in and denervated D. opalescens. (A) Activity response (means±s.e.m.) of seawater; white symbols represent responses in seawater containing chromatophores in D. gigas evoked by electrical stimulation 5 V above −1 200 nmol l TTX. (B) Analogous results with intact Doryteuthis opalescens threshold (n=15). Black symbols represent responses in seawater; white −1 and 400 nmol l TTX (n=20). We assume the small residual myogenic symbols represent responses conducted in seawater containing 2.5 µmol l−1 response is due to incomplete penetration of TTX into the tissue. 5-HT. (B) Analogous results with D. opalescens (n=25). (C) Spontaneous (C) Spontaneous activity in denervated D. gigas before (solid line) and after activity in denervated D. gigas before (solid line) and after (dashed line) −1 (dashed line) application of 200 nmol l TTX. (D) Spontaneous activity in application of 2.5 µmol l−1 5-HT. (D) Spontaneous activity in denervated denervated D. opalescens before (solid line) and after (dashed line) application D. opalescens before (solid line) and after (dashed line) application of −1 of 400 nmol l TTX. 2.5 µmol l−1 5-HT.

In both species, application of 2.5 µmol l−1 5-HT leads to spontaneous wave activity (not illustrated). Effects of the relaxation of any chromatophores that were tonically active and a pharmacological agents tested on the overall temporal variation of pallid appearance of the skin (not illustrated). In D. gigas, 5-HT spontaneous activity were quantified by calculating the variance of greatly reduces responses to electrical stimulation (Fig. 4A) but has summed chromatophore activity during a 30 s observation, and only no detectable effect on stimulated activity in intact D. opalescens 5-HT has a significant inhibitory effect in either species (Fig. 5). (Fig. 4B). Spontaneous activity in both D. gigas (Fig. 4C) and denervated D. opalescens (Fig. 4D) is essentially eliminated by the Immunohistochemistry of neurotransmitters same concentration of 5-HT. Preliminary studies on D. gigas with Samples from D. gigas contained axons labeled by anti-5-HT lower concentrations of 5-HT suggest that spontaneous activity is (Fig. 6A) and anti-L-Glu (Fig. 6B) that were associated with radial more sensitive than stimulated activity. In these studies spontaneous muscle fibers in all material examined. Both glutamatergic and activity was eliminated by 0.9 µmol l−1 5-HT (n=3), but stimulated serotonergic axons tend to run obliquely with respect to muscle activity was reduced by only 12.8% (n=15). fibers in their proximal region, with an average length of potential In all preparations of D. opalescens and D. gigas skin, bath contact between an axon and a muscle fiber of 12±15 µm (mean±s.d., application of L-Glu leads to tonic expansion of most n=263 muscle fibers). The discrete nature of the labeling and low chromatophores, but it has no obvious effect on the transient background, in conjunction with control experiments (see Materials responses of relaxed chromatophores to electrical stimulation or on and methods), suggest that the primary antibodies used are highly Journal of Experimental Biology

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A muscle fiber, consistent with previous findings (Messenger et al., 1.5 1997). The length of potential contact between a serotonergic axon and a muscle fiber is highly variable, with an average of 95± 65 µmol l−1 (n=316 muscle fibers) and therefore much longer than 10 1.0

10 in D. gigas. In denervated samples there were no detectable ϫ serotonergic axons associated with radial muscle fibers (Fig. 7B and Table 1). Glutamatergic axons were also frequently observed in 0.5 association with radial muscle fibers (Fig. 7C), and glutamatergic

Variance Variance axons were also eliminated by the denervation procedure (Fig. 7D

(pixel values squared) and Table 1). These findings confirm that transecting the pallial 0 Seawater TTX 5-HT L-Glu nerve results in complete degeneration of both glutamatergic motor axons and serotonergic axons associated with the chromatophore B network. 2.5 Double-labeling of 5-HT and L-Glu in both D. gigas and D. opalescens reveals the presence of glutamatergic axons that do 2.0 not appear to contain 5-HT, but the small diameter of these axons

11 complicates analysis by confocal microscopy. It was often difficult 10 1.5

ϫ to discern definitively whether 5-HT and L-Glu are colocalized in the same axon (Fig. 8A,B) because the axons run tightly wrapped 1.0 around each other. However, when 5-HT and L-Glu labeling are viewed separately in the same sample, there are cases in which Variance Variance 0.5 glutamatergic axons exist that do not appear to contain 5-HT (pixel values squared) – 0 (Fig. 8C F). Although this does not rule out the possibility that a Seawater TTX 5-HT L-Glu subset of axons contains both 5-HT and L-Glu, it strongly suggests that some, and probably most, glutamatergic axons do not contain 5-HT. Fig. 5. Effects of TTX, 5-HT and L-glutamate (L-Glu) on wave activity. (A) Variance of spontaneous chromatophore activity over 30 s sampling periods in skin samples from D. gigas in 200 nmol l−1 TTX (n=6), 2.5 µmol l−1 Muscle fiber morphology 5-HT (n=5) or 200 µmol l−1 L-Glu (n=5) compared with the seawater control Labeling of actin filaments with phalloidin reveals prominent (n=16). Bars represent ±1 s.e.m. 5-HT is the only treatment with an obvious branching at the distal ends of the muscle fibers in D. opalescens effect (Welch’s t-test, P=0.1008). (B) Analogous data from denervated skin (circled regions in Fig. 9A), as previously described in this species samples of D. opalescens in seawater (n=16), 400 nmol l−1 TTX (n=6), 2.5 µmol l−1 5-HT (n=5) or 100 µmol l−1 L-Glu (n=5). Bars represent ±1 s.e.m. Again, 5-HT is the only treatment with an obvious effect (Welch’s t-test, AB P=0.0442). specific. Thus, both endogenous 5-HT and L-Glu are contained in axons within the chromatophore layer, and the pharmacological data presented above are consistent with L-Glu being an excitatory neurotransmitter and 5-HT being either an inhibitory neurotransmitter or neuromodulator. In samples from intact D. opalescens, serotonergic axons are frequently associated with radial muscle fibers (Fig. 7A), with 50 µm 50 µm labeling by anti-5-HT occurring over the proximal region of the C D AB

50 µm 50 µm

50 µm50 µm Fig. 7. Serotonergic and glutamatergic axons associated with chromatophores in D. opalescens and their loss following chronic Fig. 6. Confocal images of the chromatophore layer in D. gigas. F-actin in denervation. Labeling is for F-actin (), anti-5-HT (red), anti-glutamate radial muscle fibers is labeled with phalloidin (green). Axons are labeled with (white) and nuclei () imaged at 20× using confocal microscopy. (A) Radial anti-5-HT (red) or anti-glutamate (white). Nuclei are labeled with DAPI (blue). muscle fibers and their associated serotonergic axons in intact skin. (B) Radial (A) Serotonergic axons run in a generally oblique direction with respect to muscle fibers in denervated skin labeled with phalloidin and anti-5-HT. radial muscle fibers. (B) Glutamatergic axons follow a similar course relative to Serotonergic axons are absent. (C) Radial muscle fibers and their associated muscle fibers in the same sample. These images were created by stitching glutamatergic axons in intact skin. (D) Radial muscle fibers in denervated skin together multiple images taken with a 20× objective. labeled with phalloidin and anti-glutamate. Glutamatergic axons are absent. Journal of Experimental Biology

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Table 1. Effect of chronic denervation on motor axons in the AB chromatophore layer of Doryteuthis opalescens No. muscle No. Muscle fibers with Sample fibers axons associated axons (%) Intact Serotonergic 1625 604 37.2 innervation Glutamatergic 912 653 71.6 innervation 20 µm 20 µm Denervated CD Serotonergic 1437 0 0.0 innervation Glutamatergic 817 3 0.4 innervation Numbers of radial muscle fibers examined, numbers of axons associated with a radial muscle fiber and the percentage of radial muscle fibers with an associated serotonergic or glutamatergic axon are indicated for all intact and denervated skin samples of Doryteuthis opalescens. 20 µm 20 µm

(Florey and Kreibel, 1969) and in Doryteuthis pealei (Bell et al., EF 2013). Similar distal branching also occurs in D. gigas (circles in Fig. 9B) and, in many cases, radial muscle fibers from different chromatophores appear to be connected through their distal ends (arrowhead in Fig. 9B). Examination of sequential optical sections (z-stacks) of 3 µm thickness showed that these muscle fibers do not appear to simply overlap, but rather intersect within the same plane. A total of 1037 muscle fibers in D. gigas (four squid) were 20 µm 20 µm examined and, of those, 8.0±2.3% (mean±s.d.) had a possible connection of this sort with a muscle fiber belonging to a different Fig. 8. Co-labeling of 5-HT and L-Glu in axons associated with radial chromatophore. In most cases the two chromatophores were not muscle fibers imaged using confocal microscopy. 5-HT is red, L-Glu is white and radial muscle fibers are blue/green; nuclei are labeled with DAPI directly adjacent but were separated by a third chromatophore. (blue). The images represent a single tile taken with a 40× oil objective. Putative connections of this sort are rare in D. opalescens. (A) A nerve branch in D. gigas contains closely apposed glutamatergic and Examination of 1625 muscle fibers in six samples of innervated skin serotonergic axons and runs obliquely to several radial muscle fibers. showed that only 0.3±0.4% of radial muscle fibers appeared to be (B) A nerve in D. opalescens containing both types of axons makes close connected. In denervated skin this figure was higher (0.8±0.8%, contact with a radial muscle fiber in the center of the image. (C) The image from n=1437 in six samples), but the difference between denervated and panel A with the channel used for visualizing L-Glu turned off. (D) The image from panel A with the channel used for visualizing 5-HT turned off. Although innervated skin is not significant (Wilcoxon rank-sum test, P=0.21). both serotonergic axons and glutamatergic axons are contained within the It again appears that connected chromatophores may not be directly same nerve branch, glutamatergic axons are clearly visible that are free of adjacent to one another, suggesting that connections may primarily 5-HT labeling. (E) The image from panel B with the channel for L-Glu turned off. occur between chromatophores of the same color class (or size class (F) The image from panel B shown with the channel for 5-HT turned off. in the case of D. gigas). Comparison of panels E and F shows that most glutamatergic axons do not show 5-HT. DISCUSSION This study examines the structure and function of chromatophores in synapses distributed along the proximal region of the muscle fiber Dosidicus gigas, an oceanic ommastrephid squid, and compares results (Florey and Kreibel, 1969; Reed, 1995a; Messenger et al., 1997). to parallel experiments on both intact and denervated Doryteuthis This differs from the situation in D. gigas, where both glutamatergic opalescens, a coastal loliginid species. We find that, as in loliginid and serotonergic axons pass more obliquely across muscle fibers. In squid, L-Glu and 5-HT are contained in axons associated with the this case, both types of axons would be able to form at most only a chromatophore network in D. gigas, with L-Glu probably functioning few en passant synapses with any given muscle fiber, in contrast to as an excitatory neurotransmitter and 5-HT as an inhibitory modulator the multiple neuromuscular junctions in loliginids. (or conceivably neurotransmitter). Although the roles of these The pattern of motor innervation in D. gigas is actually more compounds are similar in these two squid families, some major similar to that reported in the octopus Eledone cirrhosa (Dubas, differences in the pattern of innervation of chromatophores and in 1987). Spatial–temporal patterning by octopus chromatophores potential connectivity between chromatophores merit consideration. involves coordinated wave activity (see figure 15 of Packard and Our results suggest that 5-HT plays an important neuro-modulatory Sanders, 1971), and these animals also have a sparser innervation. role in inhibiting spontaneous chromatophore activity, but to a varying Additional comparative studies of structure and function in the degree in the two species. chromatophore network of additional species would be extremely worthwhile in furthering our understanding of the control Innervation of the chromatophore layer in D. gigas of chromogenic behaviors in these animals. Striking differences exist in the innervation pattern of the chromatophore layer of D. gigas compared to that in loliginid Myogenic chromatophore activity? squid. Our results on D. opalescens confirm that motor axons in Prominent wave activity through the chromatophore network is loliginids run directly apposed to radial muscle fibers, with multiple readily apparent in the intact skin of D. gigas and in denervated D. Journal of Experimental Biology

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Fig. 9. Labeling of radial muscle fibers. Radial muscle fibers were labeled with phalloidin (green) and nuclei with DAPI (blue). (A) Extensive branching along the distal end of a radial muscle fiber (circle) of D. opalescens. (B) Radial muscle fibers of D. gigas also show distal branching (circles). About 8% of radial muscle fibers in this species appear to be connected at their distal end to a muscle fiber from a nearby chromatophore (arrowhead). (C) Putative connections between distal branches of muscle fibers of different chromatophores in D. opalescens. Connections in this species were much rarer than those in D. gigas. These images were created by stitching together multiple images taken with a 20× objective. opalescens. In D. gigas, these waves are associated with a sparse (Rojas and Armstrong, 1971; Brismar and Gilly, 1987; Rosenthal innervation pattern and, in D. opalescens, they are accompanied by and Bezanilla, 2002; Rosenthal and Gilly, 2003). Such channels do a complete degeneration of both serotonergic and glutamatergic occur in gastropod molluscs (Gilly et al., 1997), however, and axons associated with the radial muscle fibers. In both species these cannot be entirely ruled out. waves were not affected by TTX at concentrations that greatly We found no evidence of any residual neural plexus containing reduce electrically stimulated activity. This observation confirms L-Glu or 5-HT in the skin of denervated D. opalescens.Wehavenot that these waves are not controlled by the known neural control successfully carried out immunohistochemical studies that pathway that descends from the brain. TTX-resistant sodium specifically label all neurons, so other neuronal processes could channels have not been reported in either D. gigas or loliginid have remained after denervation and escaped detection. However, species based on a large body of work on giant axons and on cell no other endogenous neurotransmitters have been identified in the bodies of the giant fiber lobe neurons that give rise to these axons chromatophore network in loliginid squid (Messenger et al., 1997; Journal of Experimental Biology

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Messenger, 2001) and no such nerve net has been found in either Preliminary recordings using whole-cell patch-clamp methods have classical histological studies (Florey, 1966) or in conjunction with not revealed any qualitative change in electrical properties of tubulin-labeling of the chromatophore layer in D. opalescens chromatophore muscle fibers following chronic denervation in hatchlings (Mackie, 2008). Therefore, the existence of a peripheral D. opalescens (W.F.G., unpublished data). Although changes neural plexus is highly unlikely, and we propose that TTX-resistant in voltage-dependent Ca2+ and/or K+ currents cannot be ruled out wave activity is most likely myogenic. at this time, Na2+ currents are not observed. Electrical properties of radial muscle fibers in D. gigas are unknown, but they might show Connectivity between chromatophores enhanced excitability under normal conditions, and such a feature Our results suggest the existence of a physical connection between would be consistent with the relative paucity of glutamatergic the branching distal ends of radial muscle fibers from different neuromuscular contacts in this species. chromatophores in both species of squid studied. Previous descriptions of direct connections between chromatophore muscle Role of serotonergic innervation fibers in other cephalopods (Froesch-Gaetzi and Froesch, 1977; Electrically stimulated responses and spontaneous chromatophore Packard, 1995a) were largely dismissed after intracellular injections waves in D. gigas are both inhibited by 5-HT, but only wave activity of Lucifer failed to reveal any such connections in in denervated D. opalescens was inhibited by this agent. We L. vulgaris (Reed, 1995b). Although these experiments confirmed propose that the extensive serotonergic innervation of the intercellular coupling between the nucleated basal regions of muscle chromatophore network in intact D. opalescens is responsible for fibers (Florey and Kreibel, 1969), they failed to reveal the distal the lack of spontaneous activity but does not interfere with branching of radial muscle fibers that is clearly evident in both electrically stimulated excitation via motor axons. This suggests D. opalescens (Florey and Kreibel, 1969; present study) and D. pealei that denervated skin in D. opalescens may be similar to the normal (Bell et al., 2013), species that are closely related to L. vulgaris. situation in D. gigas, where limited contacts between serotonergic Although visualization using confocal microscopy alone cannot axons and radial muscle fibers suggest that serotonergic inhibition is prove the existence of a physical connection between muscle fiber relatively weak, thereby facilitating a higher level of spontaneous processes (rather than a simple region of apposition) or elucidate the activity. structural nature of the connection, we found evidence of putative Mechanisms of peripheral inhibition by 5-HT are not clear in morphological connections in 8% of radial muscle fibers in D. gigas either case. Intracellular recordings in D. opalescens did not reveal but in only 0.3% of muscle fibers in D. opalescens. The much larger any effects of 5-HT on electrical properties of radial muscle fibers or fraction in D. gigas is consistent with the propagation of waves in on postsynaptic potentials (Florey and Kreibel, 1969), nor did 5-HT fresh skin preparations and potentially in the living animal in have significant effects on the rates of contraction and relaxation conjunction with flickering behavior (Rosen et al., 2015). Waves following nerve stimulation in fresh skin preparations. These occur in D. opalescens only after denervation of chromatophores, observations led to the proposal that 5-HT acts intracellularly in and this will be discussed below in regard to the loss of serotonergic radial muscle fibers to reduce the rise in intracellular Ca2+ necessary inhibition. for muscle contraction (Florey and Kreibel, 1969; Lima et al., 1997, Transmission across a distal connection between radial muscle 1998). Serotonergic inhibition could occur at any of several levels fibers might involve intercellular junctions through which ions or between release of L-Glu from motor axons and the rise in small molecules can move (e.g. gap junctions), a physical intracellular Ca2+ in radial muscle fibers, i.e. the processes that connection through which mechanical activity of neighboring define excitation–contraction coupling. In the case of myogenic muscle fibers could be sensed by stretch-activated channels, or activity, effects on excitability might also be relevant. Further some other mechanism. Wave activity described in this study discussion of mechanisms for serotonergic modulation of propagates at a velocity of ∼1cms−1, at least an order of magnitude chromatophore activity in D. gigas or loliginid squid must be faster than intercellular Ca2+ waves in a large variety of tissues considered speculative at this time. (Jaffe, 1991; Haas et al., 2006), and this discrepancy strongly Despite uncertainty over mechanisms, serotonergic inhibition suggests that Ca2+ waves alone cannot account for propagation of probably plays an important role in chromogenic behaviors of the waves through the chromatophore network of squid. living squid. Stimulated activity was unaffected by 5-HT in Mechanisms responsible for the initiation of chromatophore D. opalescens and there was some indication in our experiments waves are also unknown, but excitability properties of radial muscle that spontaneous activity in D. gigas is more readily suppressed by fibers would be relevant. These cells do not show evidence of 5-HT than is stimulated activity. A significant difference in voltage-gated sodium channels in D. opalescens (Florey and sensitivity would imply that endogenous 5-HT in the skin could Kreibel, 1969), but they do have voltage-gated calcium channels provide inhibitory control over spontaneous chromogenic activity that provide a graded type of excitability that can support spikes that without interfering with neutrally driven activity. In D. opalescens, arise from generator potentials during spontaneous pulsations of inhibition of spontaneous wave activity might be particularly single chromatophores (Florey, 1966). Such pulsations do not important, because this species relies heavily on spatially well- propagate, but it seems likely that a similar form of electrogenesis defined displays for both and communication. Any would be involved in initiating waves. significant degree of spontaneous chromatophore activity, Vertebrate skeletal muscle fibers can become spontaneously particularly wave-type activity that characterizes the denervated active after denervation due to changes in density and properties of condition, would seemingly disrupt these displays. Conversely, several types of ion channels (Harris and Thesleff, 1971; Miledi dynamic chromogenic displays are common to the repertoire of et al., 1971; Pappone, 1980; Caldwell and Milton, 1988; Neelands D. gigas (Rosen et al., 2015), and we propose that flickering et al., 2001; Midrio, 2006). Comparable studies do not appear to behavior in the living squid represents a form of wave activity with have been reported for invertebrate muscle, but excitability of radial weak serotonergic inhibition acting to keep flickering in a balanced muscle fibers in D. opalescens could be enhanced post-denervation, state. Stronger and global inhibitory control of flickering is also thereby facilitating initiation and propagation of myogenic activity. possible in this species, as demonstrated by the squid’s ability to Journal of Experimental Biology

4678 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 4669-4680 doi:10.1242/jeb.164160 rapidly halt this ongoing behavior, often in conjunction with the 9366-13 (National Geographic Society) to W.F.G., and by Young Explorer Grant onset of other chromogenic displays, particularly flashing. It seems 9424-14 (National Geographic Society) to H.E.R. Funding for the confocal microscopy facility was provided by US National Science Foundation grant likely that 5-HT would also underlie this stronger inhibition via FSML 122726. descending control through the serotonergic innervation. Supplementary information Vertical versus horizontal control of chromogenic behaviors Supplementary information available online at Coordinated activity in the chromatophore network that is not http://jeb.biologists.org/lookup/doi/10.1242/jeb.164160.supplemental directly driven by descending (vertical) motor control was References previously identified as a distributed, horizontal control process Bell, G. R. R., Kuzirian, A. M., Senft, S. L., Mäthger, L. M., Wardill, T. J. and (Packard, 2001, 2006). Results of the present study support this Hanlon, R. T. (2013). Chromatophore radial muscle fibers anchor in flexible squid idea. Spontaneous waves of chromatophore activity are common in skin. Invertebr. Biol. 132, 120-132. Bone, Q. and Howarth, J. V. (1980). The role of L-glutamate in neuromuscular D. gigas, and this activity propagates via a TTX-resistant pathway transmission in some molluscs. J. Mar. Biol. Assoc. UK 60, 619-626. within the skin. Transmission may involve the putative connections Brismar, T. and Gilly, W. F. (1987). Synthesis of sodium channels in the cell bodies between the branching distal ends of radial muscle fibers, although of squid giant axons. Proc. Natl. Acad. Sci. USA 84, 1459-1463. the mechanism of coupling remains unknown. Nevertheless, such a Burford, B. P., Robison, B. H. and Sherlock, R. E. (2014). Behaviour and in the juvenile and subadult life stages of the mesopelagic squid Chiroteuthis horizontal control system might coordinate chromogenic activity in calyx. J. Mar. Biol. Assoc. UK 95, 1221-1235. the living squid with minimal, or even no, descending vertical Bush, S. L., Robison, B. H. and Caldwell, R. L. (2009). Behaving in the dark: control. locomotor, chromatic, postural, and bioluminescent behaviors of the deep-sea Waves of chromatophore activity are not evident in living squid Octopoteuthis deletron Young 1972. Biol. Bull. 216, 7-22. Caldwell, J. H. and Milton, R. L. (1988). Sodium channel distribution in normal and D. opalescens or in fresh skin preparations, but such activity is denervated rodent and snake skeletal muscle. J. Physiol. 401, 145-161. clearly present in denervated animals. Our comparative approach Cornwell, C. J. and Messenger, J. B. (1995). Neurotransmitters of squid suggests that, if horizontal control is operative in intact chromatophores. In Cephalopod Neurobiology: Neuroscience Studies in Squid, Octopus, and Cuttlefish (ed. N. J. Abbott, R. Williamson and L. Maddock), pp. D. opalescens, it must be much less well developed and/or more 369-379. Oxford, UK: Oxford University Press. strongly inhibited than it is in D. gigas. Given the similarities in the Dubas, F. (1987). Innervation of chromatophore muscle fibres in the octopus chromatophore system between the two distantly related species of Eledone cirrhosa. Cell Tissue Res. 248, 675-682. squid studied (i.e. different families), we propose that horizontal Florey, E. (1966). Nervous control and spontaneous activity of the chromatophores of a cephalopod, Loligo opalescens. Comp. Biochem. Physiol. 18, 305-324. control is common to all species of coleoid cephalopods, but the Florey, E. and Kreibel, M. E. (1969). Electrical and mechanical responses of extent to which it is involved in natural chromogenic behaviors will chromatophore muscle fibers of the squid, Loligo opalescens, to nerve stimulation depend on the precise needs of a particular species in the context of and drugs. Z. Vergl. Physiol. 65, 98-130. its environment. Florey, E., Dubas, F. and Hanlon, R. T. (1985). Evidence for L-glutamate as a transmitter substance of motoneurons innervating squid chromatophore muscles. How horizontal and vertical processes coordinate to control Comp. Biochem. Physiol. C. 82, 259-268. chromogenic behaviors in real time remains to be elucidated for any Froesch-Gaetzi, V. and Froesch, D. (1977). Evidence that chromatophores of specific case, but this interaction is evident from a different perspective. cephalopods are linked by their muscles. Experientia 33, 1448-1450. Gilly, W. F., Gillette, R. and McFarlane, M. (1997). Fast and slow activation kinetics Position, resting size and color of individual chromatophores making of voltage-gated sodium channels in molluscan neurons. J. Neurophysiol. 77, up the network are the result of developmental history (Packard, 1985), 2373-2384. itself a hierarchical, vertical process, and waves of chromatophore Hanlon, R. T. and Messenger, J. B. (1996). Cephalopod Behavior. Cambridge: activity tend to involve chromatophores of the same size/color class Cambridge University Press. Hannon, J. and Hoyer, D. (2008). Molecular biology of 5-HT receptors. Behav. (Packard, 2001; http://gillylab.stanford.edu/assets/packard/ap_vid1_ Brain Res. 195, 198-213. 320x240.ogv). This clearly provides a manifestation of vertical and Harris, J. B. and Thesleff, S. (1971). Studies on tetrodotoxin resistant action horizontal control working in concert. potentials in denervated skeletal muscle. Acta Physiol. Scand. 83, 382-388. Haas, J. S., Nowotny, T. and Abarbanel, H. D. I. (2006). Spike-timing-dependent plasticity of inhibitory synapses in the entorhinal cortex. J. Neurophysiol. 96, Acknowledgements 3305-3313. We thank Patrick Daniel for technical assistance and photography of squid on the R/ Hofmann, F. B. (1907). Gibt es in der Muskulatur der Mollusken periphere, V Puma at the invitation of Dr Carlos Robinson, UNAM, Mexico City; Diana Li, Elan kontinuierlich leitende Nervennetze bei Abwesenheit von Ganglienzellen? Porter, Alex Norton and Joe Welsh (Monterey Bay Aquarium) for squid collection; I. Untersuchungen an Cephalopoden. Archiv Gesamte Physiol. 118, 375-412. Russel Williams (Hopkins Marine Station) for programming assistance; Leonel Hoving, H.-J. T., Gilly, W. F., Markaida, U., Benoit-, K. J., West-Brown, Z., Orozco and staff at Minera Metallurgica Boleo, for general support; Karmina Arroyo Daniel, P., Field, J. C., Parassenti, L., Liu, B. and Campos, B. (2013). Extreme Ramirez and Institutuo Tecnologico Superior de Mulege for assistance in the field; plasticity in life-history strategy allows a migratory predator ( jumbo squid) to cope and Dr Christopher Lowe (Hopkins Marine Station) for guidance in with a changing climate. Glob. Change Biol. 19, 2089-2103. immunohistochemistry procedures. We are particularly grateful for critical comments Jaffe, L. F. (1991). The path of calcium in cytosolic calcium oscillations: a unifying on the manuscript by Andrew Packard (research affiliate of Hopkins Marine Station). hypothesis. Proc. Natl. Acad. Sci. USA 88, 9883-9887. Kolodziejczyk, A., Sun, X., Meinertzhagen, I. A. and Nässel, D. R. (2008). Competing interests Glutamate, GABA and acetylcholine signaling components in the lamina of the The authors declare no competing or financial interests. Drosophila . PLoS ONE 3, e2110. Laan, A., Gutnick, T., Kuba, M. J. and Laurent, G. (2014). Behavioral analysis of Author contributions cuttlefish traveling waves and its implications for neural control. Curr. Biol. 24, 1737-1742. Conceptualization: H.E.R., W.F.G.; Methodology: H.E.R., W.F.G.; Software: H.E.R.; Lima, P. A., Messenger, J. B. and Brown, E. R. (1997). Monitoring cytoplasmic Validation: H.E.R.; Formal analysis: H.E.R.; Resources: W.F.G.; Writing - original Ca2+ in cephalopod chromatophore muscles. J. Physiol. 504P, P2-P3. draft: H.E.R.; Writing - review & editing: H.E.R., W.F.G.; Visualization: W.F.G.; Lima, P. A., Messenger, J. B. and Brown, E. R. (1998). 5-HT suppresses Ca2+ Supervision: W.F.G.; Project administration: W.F.G.; Funding acquisition: release from ryanodine-sensitive stores in squid chromatophore muscle. H.R., W.F.G. J. Physiol. 513P, 127P. Lima, P. A., Nardi, G. and Brown, E. R. (2003). AMPA/kainate and NMDA-like Funding glutamate receptors at the chromatophore neuromuscular junction of the squid: This work was supported by funds from Stanford University (H.E.R.) and by grants role in synaptic transmission and skin patterning. Eur. J. Neurosci. 17, 507-516. OCE-1338973 RAPID, IOS-142093 EAGER, OCE 0850839 and IOS-1557754 (US Mackie, G. O. (2008). Immunostaining of peripheral nerves and other tissues in

National Science Foundation), N000140911054 (US Office of Naval Research) and whole mount preparations from hatchling cephalopods. Tissue Cell 40, 21-29. Journal of Experimental Biology

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